A dynamic quaternary structure of bovine .alpha.-crystallin as indicated from intermolecular exchange of subunits

105

To determine the effects of deamidation on structural and functional properties of ␣A-crystallin, three mutants (N101D, N123D, and N101D/N123D) were generated. Deamidated ␣B-crystallin mutants (N78D, N146D, and N78D/N146D), characterized in a previous study (Gupta, R., and Srivastava, O. P. (2004) Invest. Ophthalmol. Vis. Sci. 45, 206 -214) were also used. The biophysical and chaperone properties were determined in (a) homoaggregates of ␣A mutants (N101D, N123D, and N101D/N123D) and (b) reconstituted heteroaggregates of ␣-crystallin containing (i) wild type ␣A (WT-␣A): WT-␣B crystallins, (ii) individual ␣A-deamidated mutants:WT-␣B crystallins, and (iii) WT-␣A:individual ␣B-deamidated mutant crystallins. Compared with the WT-␣A, the three ␣A-deamidated mutants showed reduced levels of chaperone activity, alterations in secondary and tertiary structures, and larger aggregates. These altered properties were relatively more pronounced in the mutant N101D compared with the mutant N123D. Further, compared with heteroaggregates of WT-␣A and WT-␣B, the heteroaggregates containing deamidated subunits of either ␣A-or ␣B-crystallins and their counterpart WT proteins showed higher molecular mass, altered tertiary structures, lower exposed hydrophobic surfaces, and reduced chaperone activity. However, the heteroaggregate containing WT-␣A and deamidated ␣B subunit showed lower chaperone activity, smaller oligomers, and 3-fold lower subunit exchange rate than heteroaggregate containing deamidated ␣A-and WT-␣B subunits. Together, the results suggested that (a) both Asn residues (Asn-101 and Asn-123) are required for the structural integrity and chaperone function of ␣A-crystallin and (b) the presence of WT-␣B in the ␣-crystallin heteroaggregate leads to packing-induced structural changes which influences the oligomerization and modulate chaperone activity.␣-Crystallin, the most abundant protein in lens mature fiber cells, constitutes ϳ35% of the total lens protein. In vivo, the ␣A and ␣B subunits at a ratio of 3:1 form an oligomer of 800 kDa. Both ␣A-and ␣B-crystallins are small heat shock proteins (Hsps), 1 and show molecular chaperone activity to protect proteins from aggregation in the eye lens (1, 2). Because of this property, ␣-crystallin is believed to play a crucial role in maintaining the lens transparency. Like other small heat shock proteins, ␣-crystallin also contains a highly conserved sequence of 80 -100 residues (residues 62-143 in ␣A-and 66 -147 in ␣B-crystallin) called the ␣-crystallin domain (3, 4). Based on similarities with the structure of other Hsps, it is believed that the N-terminal region (residues 1-62 in ␣A-and 1-66 in ␣B-crystallin(s)) of ␣-crystallin forms an independently folded domain, whereas the C terminus (referred as the C-terminal extension; residues 143-173 in ␣A-and 147-175 in ␣B-crystallin) is flexible and unstructured (4). Previous reports show that the removal of N-terminal residues (56 residues) and C-terminal extensions (32-34 residues) of ␣A-and ␣B-crystallins lead to improper folding,...

Section: Discussionmentioning

confidence: 99%

Deamidation Affects Structural and Functional Properties of Human αA-Crystallin and Its Oligomerization with αB-Crystallin

Gupta

Srivastava

2004

105

“…in urea or guanidine HCl), and the ␣A-and ␣B-crystallins were reconstituted by removal of the denaturant (23,(37)(38)(39). The molecular weights of these reconstituted ␣-crystallins were always lower than that of native ␣-crystallin, indicating that the reaggregation is incomplete.…”

Section: Discussionmentioning

confidence: 99%

Conformational and Functional Differences between Recombinant Human Lens αA- and αB-Crystallin

Sun

Das

Liang

1997

158

136

Human and other mammalian lens proteins are composed of three major crystallins: ␣-, ␤-, and ␥-crystallin. ␣-Crystallin plays a prominent role in the supramolecular assembly required to maintain lens transparency. With age, the crystallins, especially ␣-crystallin, undergo posttranslational modifications that may disrupt the supramolecular assembly, and the lens becomes susceptible to other stresses resulting in cataract formation. Because these modifications occur even at a relatively young age, it is difficult to obtain pure, unmodified crystallins for in vitro experiments. ␣-Crystallin is composed of two subunits, ␣A and ␣B. Before the application of recombinant DNA technology, these two ␣-crystallin subunits were separated from calf lens in the denatured state and reconstituted by the removal of the denaturant, but they were not refolded properly. In the present studies, we applied the recombinant DNA technology to prepare native, unmodified ␣A-and ␣B-crystallins for conformational and functional studies. The expressed proteins from Escherichia coli are in the native state and can be studied directly. First, ␣A and ␣B cDNAs were isolated from a human lens epithelial cell cDNA library. The cDNAs were cloned into a pAED4 expression vector and then expressed in E. coli strain BL21(DE3). Pure recombinant ␣A-and ␣B-crystallins were obtained after purification by gel filtration and DEAE liquid chromatography. They were subjected to conformational studies involving various spectroscopic measurements and an assessment of chaperone-like activity. ␣A-and ␣B-crystallins have not only different secondary structure, but also tertiary structure. 1-Anilino-8-naphthalene sulfonate fluorescence indicates that ␣B-crystallin is more hydrophobic than ␣A-crystallin. The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for ␣B-than for ␣A-crystallin. The resulting data provide a base line for further studies of human lens ␣-crystallin.␣-Crystallin is the major lens structural protein responsible for the supramolecular assembly necessary to maintain lens transparency. It is isolated as a large water-soluble aggregate with a mass of 800 kDa and is composed of two homologous subunits, ␣A and ␣B, each with a molecular mass of 20 kDa. With aging and cataract formation, ␣-crystallins gradually become a high molecular weight aggregate and eventually an insoluble protein (1). This is manifested by the observation that the major crystallin component in the insoluble protein fraction of aged and cataractous lenses is ␣-crystallin (2). The aggregation and insolubilization of ␣-crystallin are believed to arise from posttranslational modifications, which accumulate significantly because of the low turnover rate of lens crystallins. Two-dimensional gel electrophoresis indicated that human lens crystallins were modified extensively even at a relatively young age (3). The significance of the modification is that crystallins become partially unfolded, and this partially unfolding exposes ...

“…Separation of ␣A-and ␣B-crystallins and Generation of Homoaggregates-The subunits of ␣-crystallin can be separated by a variety of methods (11,27,28). The subunits were separated on a C4 reverse phase column using a water-acetonitrile gradient containing 0.08% trifluoroacetic acid.…”

Section: Methodsmentioning

confidence: 99%

Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Datta

Rao

1999

104

␣-Crystallin, a heteromultimeric protein made up of ␣A-and ␣B-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of ␣-crystallin can enhance its chaperone-like activity severalfold. The two subunits of ␣-crystallin have extensive sequence homology and individually display chaperonelike activity. We have investigated the chaperone-like activity of ␣A-and ␣B-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, ␣B-crystallin shows significant protective ability even at subphysiological temperatures, at which ␣A-crystallin or heteromultimeric ␣-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of ␤ L -crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quenching by acrylamide shows that the tryptophans in ␣B-crystallin are more accessible than the lone tryptophan in ␣A-crystallin even at 25°C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on ␣B-crystallin. Circular dichroism studies show some tertiary structural changes in ␣A-crystallin above 50°C. ␣B-crystallin, on the other hand, shows significant alteration of tertiary structure by 45°C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, ␣B-crystallin is much more sensitive to temperaturedependent structural perturbation than ␣A-or ␣-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of ␣-crystallin and indicate different roles for the two proteins both in ␣-crystallin heteroaggregate and as separate proteins under stress conditions. ␣-Crystallin is a major protein of the mammalian lens and constitutes as much as 50% of its dry weight. Studies over the past few years have shown that ␣-crystallin is expressed in several nonlenticular tissues such as heart, brain, and kidney, and its expression is enhanced severalfold during stress and disease conditions (1-6). ␣-Crystallin is shown to have homology with small heat shock proteins (7-10). Horwitz (11) shows that ␣-crystallin can prevent the thermal aggregation of ␤-and ␥-crystallins and a few other proteins like a molecular chaperone. Demonstration of chaperone-like activity of ␣-crystallin has provided an excellent opportunity to investigate the mechanistic aspects of chaperone function in general and the role of ␣-crystallin under stress conditions in particular. It is possible that, in the lens, ␣-crystallin may chaperone the formation of the transparent and appropriately ...